Biology subjects

den Ridder, M.

Publications and source records attributed to den Ridder, M..

4 recordsLinked to original sources

bioRxiv2022

Proteome dynamics during transition from exponential to stationary phase under aerobic and anaerobic conditions in yeast

The yeast Saccharomyces cerevisiae is a widely used eukaryotic model organism and a promising cell factory for industry. However, despite decades of research, the regulation of its metabolism is not yet fully understood, and its complexity represents a major challenge for engineering and optimising biosynthetic routes. Recent studies have demonstrated the potential of resource and proteomic allocation data in enhancing models for metabolic processes. However, comprehensive and accurate proteome dynamics data that can be used for such approaches are still very limited. Therefore, we performed a quantitative proteome dynamics study to comprehensively cover the transition from exponential to stationary phase for both aerobically and anaerobically grown yeast cells. The combination of highly controlled reactor experiments, biological replicates and standardised sample preparation procedures ensured reproducibility and accuracy. Additionally, we selected the CEN.PK lineage for our experiments because of its relevance for both fundamental and applied research. Together with the prototrophic, standard haploid strain CEN.PK113-7D, we also investigated an engineered strain with genetic minimisation of the glycolytic pathway, resulting in the quantitative assessment of over 1700 proteins across 54 proteomes. These proteins account for nearly 40% of the overall yeast proteome and approximately 99% of the total protein biomass. The anaerobic cultures showed remarkably less proteome-level changes compared to the aerobic cultures, during transition from the exponential to the stationary phase as a consequence of the lack of the diauxic shift in the absence of oxygen. These results support the notion that anaerobically growing cells lack time and resources to adapt to changes in the environment. This proteome dynamics study constitutes an important step towards better understanding of the impact of glucose exhaustion and oxygen on the complex proteome allocation process in yeast. Finally, the established proteome dynamics data provide a valuable resource for the development of resource allocation models as well as for metabolic engineering efforts.

systems biology↗

bioRxiv2022

Extracting and characterizing protein-free megabasepair DNA for in vitro experiments

Chromosome structure and function is studied in cells using imaging and chromosome-conformation-based methods as well as in vitro with a range of single-molecule techniques. Here we present a method to obtain genome-size (megabasepair length) deproteinated DNA for in vitro studies, which provides DNA substrates that are two orders of magnitude longer than typically studied in single-molecule experiments. We isolated chromosomes from bacterial cells and enzymatically digested the native proteins. Mass spectrometry indicated that 97-100% of DNA-binding proteins are removed from the sample. Upon protein removal, we observed an increase in the radius of gyration of the DNA polymers, while quantification of the fluorescence intensities showed that the length of the DNA objects remained megabasepair sized. In first proof-of-concept experiments using these deproteinated long DNA molecules, we observed DNA compaction upon adding the DNA-binding protein Fis or PEG crowding agents and showed that it is possible to track the motion of a fluorescently labelled DNA locus. These results indicate the practical feasibility of a genome-in-a-box approach to study chromosome organization from the bottom up.

biophysics↗

bioRxiv2022

A systematic evaluation of yeast sample preparation protocols for spectral identifications, proteome coverage and post-isolation modifications

The importance of obtaining comprehensive and accurate information from cellular proteomics experiments asks for a systematic investigation of sample preparation protocols, particularly when working with unicellular organisms with strong cell walls, such as found in the model organism and cell factory S. cerevisiae. Sample preparation protocols may bias towards specific protein fractions or challenge the analysis of native protein modifications due to reagent-induced artefacts. Here, we performed a systematic comparison of sample preparation protocols using a matrix of different conditions commonly applied in whole cell lysate proteomics. The different protocols were evaluated for their overall fraction of identified spectra, proteome and amino acid sequence coverage, GO-term distribution and number of peptide modifications, by employing a combination of database and unrestricted modification search approaches. The best proteome and amino acid sequence coverage was achieved by using Urea combined with filter-aided or in-solution digestion protocols, where the overall outcomes were strongly influenced by the employed quenching procedure. Most importantly, the use of moderate incubation temperatures and times, circumvented excessive formation of modification artefacts. Extensive reagent-induced peptide modifications, however, were observed when using solvents such as acetone or additives such as formic acid. Moreover, several filter material-related modifications were observed when employing the filter-aided procedures. Ultimately, the best protocols enabled the identification of approximately 65-70% of all acquired fragmentation spectra, where additional de novo sequencing suggests that unidentified spectra were largely of too low spectral quality to provide confident spectrum matches. This study demonstrates the large impact of different sample preparation procedures on the proteomic analysis outcome, where the collected protocols and large sets of associated mass spectrometric raw data provide a resource to evaluate and design new protocols and guide the analysis of (native) peptide modifications in the model eukaryote yeast.

systems biology↗

bioRxiv2021

A yeast with muscle does not run faster: full humanization of the glycolytic pathway in Saccharomyces cerevisiae

While transplantation of single genes in yeast plays a key role in elucidating gene functionality in metazoans, technical challenges hamper the humanization of full pathways and processes. Empowered by advances in synthetic biology, this study demonstrates the feasibility and implementation of full humanization of glycolysis in yeast. Single gene and full pathway transplantation revealed the remarkable conservation of both glycolytic and moonlighting functions and, combined with evolutionary strategies, brought to light novel, context-dependent responses. Remarkably, human hexokinase 1 and 2, but not 4, required mutations in their catalytic or allosteric sites for functionality in yeast, while hexokinase 3 was unable to complement its yeast ortholog. Comparison with human tissues cultures showed the preservation of turnover numbers of human glycolytic enzymes in yeast and human cell cultures. This demonstration of transplantation of an entire, essential pathway paves the way to the establishment of species, tissue and disease-specific metazoan models. One Sentence SummaryThis work demonstrates the successful humanization of an entire pathway in Saccharomyces cerevisiae and establishes an attractive strategy to study (human) glycolysis architecture and regulation. HighlightsO_LIThe successful humanization of the entire glycolytic pathway in yeast offers new microbial models for both fundamental and applied studies. C_LIO_LIBoth glycolytic and moonlighting functions and turnover numbers of glycolytic enzymes are highly conserved between yeast and human. C_LIO_LIFunctionality of human hexokinases 1 and 2 in yeast requires mutations in the catalytic or allosteric binding sites. C_LIO_LICombination of single gene and full transplantation with laboratory evolution reveals context-dependent activity and evolution of glycolytic enzymes. C_LI

systems biology↗